MRG1, the product of a melanocyte-specific gene related gene, is a cytokine-inducible transcription factor with transformation activity

Abstract

Identification of cytokine-inducible genes is imperative for determining the mechanisms of cytokine action. A cytokine-inducible gene, mrg1 [melanocyte-specific gene (msg1) related gene], was identified through mRNA differential display of interleukin (IL) 9-stimulated and unstimulated mouse helper T cells. In addition to IL-9, mrg1 can be induced by other cytokines and biological stimuli, including IL-1alpha, -2, -4, -6, and -11, granulocyte/macrophage colony-stimulating factor, interferon gamma, platelet-derived growth factor, insulin, serum, and lipopolysaccharide in diverse cell types. The induction of mrg1 by these stimuli appears to be transient, with induction kinetics similar to other primary response genes, implicating its role in diverse biological processes. Deletion or point mutations of either the Box1 motif (binds Janus kinase 1) or the signal transducer and activator of transcription 3 binding site-containing region within the intracellular domain of the IL-9 receptor ligand binding subunit abolished or greatly reduced mrg1 induction by IL-9, suggesting that the Janus kinase/signal transducer and activator of transcription signaling pathway is required for mrg1 induction, at least in response to IL-9. Transfection of mrg1 cDNA into TS1, an IL-9-dependent mouse T cell line, converted these cells to IL-9-independent growth through a nonautocrine mechanism. Overexpression of mrg1 in Rat1 cells resulted in loss of cell contact inhibition, anchorage-independent growth in soft agar, and tumor formation in nude mice, demonstrating that mrg1 is a transforming gene. MRG1 is a transcriptional activator and may represent a founding member of an additional family of transcription factors.

Figure 1

mrg1 is inducible by various biological stimuli in different cell types. (A) Expression of mrg1 transcript in mouse adult tissues. (B) Induction of mrg1 expression by cytokines in the IL-2Rγ chain cytokine family. (C) mrg1 expression in various cell types induced by different biological stimuli. Total RNAs (10 μg per lane) were separated through a 1% formadehyde-agarose gel and transferred onto a nitrocellulose membrane. The same blot was sequentially hybridized with ³²P-labeled mrg1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or rRNA probes.

Figure 2

IL-9-induced mrg1 expression is mediated by JAK/STAT pathway. (A) Construction of pFlag-CMV-IL-9Rα plasmids containing wild-type (W) and mutated hIL-9Rα (18). Transmembrane domain (TM), BOX1 motif (BOX1), serine-rich region (SR), C-terminal truncation mutants deleting amino acids 422–522 (Daa422–522) or amino acids 338–522 (Daa338–522), internal deletion of BOX1 [Daa298–315(BOX1)] and point mutation within BOX1 motif (Paa306XPaa308 to Aaa306XAaa308, MBOX1) or STAT3 binding site (YLPQaa408 to YLPAaa408; MSTAT3) are as indicated. (B) Induction of mrg1 gene expression by hIL-9 in TS1 transfectants expressing different forms of hIL-9Rα. Cells were starved in serum-free medium for 8 hr and then stimulated with (+) or without (−) hIL-9 (30 ng/ml) for 1 hr. Ten micrograms of total RNA from unstimulated or stimulated cells was loaded to each lane. The same blot sequentially was hybridized with ³²P-labeled mrg1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes.

Figure 3

MRG1 is a nuclear protein with transcriptional activation function. (A) Cellular localization of MRG1 protein. pEGFP vector (CONTROL) or pMRG1-EGFP hybrid plasmid (MRG1) was transfected into NIH 3T3 by electroporation. Living cells were observed by fluorescence microscopy at 48 hr posttransfection. (B) Transcriptional activation of MRG1. NIH 3T3 cells were cotransfected with pM (negative control), pM3VP16 (positive control), GAL4BD-VP16 fusion protein), or pMMRG1 (GAL4BD-MRG1 fusion protein) and reporter plasmid pG5CAT. The assay for CAT activity was performed 48 hr posttransfection. All transfections were normalized according to β-galactosidase activity, which was used as an internal control for transfection. Data (X ± SEM) shown are representative of three independent experiments.

Figure 4

Histology of tumor in MRG1–1-injected nude mice. Photomicrograph of tumor removed from mice injected with MRG1–1 cells, and stained with hematoxylin and eosin. Histologic pattern is sarcomatoid, consistent with the connective tissue origin of the parental cells. (Magnification: ×100.)